None of the following is admitted to be prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of tyrosine residues on proteins. The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine kinases (TKs) and tyrosine phosphatases (TPs).
Receptor tyrosine kinases (RTKs) belong to a family of transmembrane proteins and have been implicated in cellular signaling pathways. The predominant biological activity of some RTKs is the stimulation of cell growth and proliferation, while other RTKs are involved in arresting growth and promoting differentiation. In some instances, a single tyrosine kinase can inhibit, or stimulate, cell proliferation depending on the cellular environment in which it is expressed.
RTKs are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. Ligand binding to membrane-bound receptors induces the formation of receptor dimers and allosteric changes that activate the intracellular kinase domains and result in the self-phosphorylation (autophosphorylation and/or transphosphorylation) of the receptor on tyrosine residues. Their intrinsic tyrosine kinase is activated upon ligand binding, thereby initiating a complex signal transduction pathway that begins with receptor autophosphorylation and culminates in the tyrosine phosphorylation of a variety of cellular substrates and ultimately in the initiation of nuclear events necessary for the overall cell response. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signaling molecules, thereby activating various signal transduction pathways.
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases do not contain a hydrophobic transmembrane domain or an extracellular domain and share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains include the SH2 domains (SRC homology domain 2) and SH3 domains (SRC homology domain 3). The non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction.
A central feature of signal transduction (for reviews, see Posada and Cooper, Mol. Biol. Cell 3:583-392, 1992; Hardie, Symp. Soc. Exp. Biol. 44:241-255, 1990), is the reversible phosphorylation of certain proteins. Receptor phosphorylation stimulates a physical association of the activated receptor with target molecules. Some of the target molecules such as phospholipase Cγ are in turn phosphorylated and activated. Such phosphorylation transmits a signal to the cytoplasm. Other target molecules are not phosphorylated, but assist in signal transmission by acting as adapter molecules for secondary signal transducer proteins. For example, receptor phosphorylation and the subsequent allosteric changes in the receptor recruit the Grb-2/SOS complex to the catalytic domain of the receptor where its proximity to the membrane allows it to activate ras.
The secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation. Reviews describing intracellular signal transduction include Aaronson, Science, 254:1146-1153, 1991; Schlessinger, Trends Biochem. Sci., 13:443-447, 1988; and Ullrich and Schlessinger, Cell, 61:203-212, 1990.
The importance of modular binding domains in regulating interactions between signaling modules, as well as their activity, is well established. The pleckstrin homology (PH) domain has been proposed to represent such a module. It contains around 120 amino acids, and was identified as a region of sequence homology, shared with pleckstrin, that appeared in a large number of proteins known to be involved in intracellular signaling.
Several studies have suggested that PH domains, especially that of the β-adrenergic receptor kinase (βARK), bind the βγ-subunits of heterotrimeric G-proteins (Gβγ). A related suggestion is that PH domains recognize the β-transducin or WD-40 repeat, found in Gβ as well as in such proteins as RACK1, a receptor for activated protein kinase C (PKC). It has also been reported that the PH domain of the non-receptor tyrosine kinase Btk interacts directly with PKC.
PH domain ligands of a different nature have also been suggested. Yoon H. S. et al., Nature 369, 672-675, 1994 argued that the N-terminal PH domain of pleckstrin bore topological resemblance to retinol binding protein (RBP), and that it might bind a similarly hydrophobic ligand. However, retinol binds to a large cavity in the hydrophobic core of RBP, and there is no such cavity in the hydrophobic core of PH domains.
The amino-terminal region of phospholipase C-δ1 (PLC-δ1), which contains a PH domain is essential for high-affinity binding of the enzyme to lipid vesicles containing PIP2. Proteolytic removal of the PLC-δ1 amino-terminal domain abolishes high affinity PIP2 binding by the enzyme, although the fragments retain catalytic activity. In common with other phospholipases, intact PLC-δ1 hydrolyzes micellar or bilayer aggregates of its substrate more effectively than it does substrate monomers. It has been suggested that the amino-terminal region of PLC-δ1 represents a noncatalytic substrate binding site that serves to absorb the enzyme to a membrane containing PIP2. Cifuentes, M. E. et al., J. Biol. Chem. 268, 11586-11593, 1993. Under these conditions, the catalytic moiety could hydrolyze substrate processively, while remaining associated with the membrane; “scooting” along the interface as described for secretory phospholipase A2 (sPLA2). Ramirez, F., and Jain, M. K., Proteins, Structure, Function and Genetics, 9:229-239, 1991.
D-myo-inositol 1,4,5-trisphosphate (I(145)P3), the head-group product of PIP2 hydrolysis by PLC, also binds to PLC-δ1, and inhibits its high-affinity binding to PIP2-containing vesicles. I(145)P3 inhibits PLC-δ1 activity in an apparently noncompetitive fashion. Removal of the amino-terminal portion of PLC-δ1, in addition to preventing high-affinity binding to PIP2, abolishes the effects of I(145)P3, indicating that PIP2 and I(145)P3 bind to the same site.
Harlan, J. E. et al., Nature 371, 168-170, 1994, have reported that several PH domains, including two from pleckstrin, as well as those from RasGAP, Tsk and βARK, can bind to vesicles containing phosphatidylinositol-(4,5)-bisphosphate (PIP2). The measured KD for binding of the N-terminal pleckstrin PH domain to PIP2 in detergent was around 30 μM.